Microsample treatment apparatus

ABSTRACT

A microsample treatment apparatus and an apparatus for detecting chemotaxis of cells and separating chemotactic cells includes a number of wells that are connected to each other via a part having resistance to fluids. The wells are each provided with tubes for injecting/sucking a sample and, if necessary, tubes for relieving pressure changes at the injection/suction. The tubes have a space in common at the top ends thereof in which a liquid can be held.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/10684 which has an Internationalfiling date of Dec. 6, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

This invention relates to an apparatus for treating liquid samples inmicroquantities. More particularly, it relates to a microsampletreatment apparatus having a structure whereby, in the step of injectinga liquid sample into a microwell for holding a sample to be reacted,analyzed, detected, etc., the overflow of the sample or the migrationthereof into another well connected thereto can be prevented and theposition of the sample in the microwell can be adjusted.

The present invention further relates to an apparatus for judgingwhether or not cells can migrate in a definite direction by their ownactions, observing the state of cells migrating in a definite directionby their own actions, or counting cells having migrated in a definitedirection by their own actions (i.e., an apparatus for detectingchemotaxis of cells). Furthermore, the present invention relates to anapparatus for separating cells based on the selective migration of cellsby their own actions. More particularly speaking, it relates to anapparatus for detecting chemotaxis of cells or separating chemotacticcells having a structure wherein, in the step of injecting a liquidsample into a microwell for holding a cell suspension or aspecimen/sample to be detected, separated, etc., the overflow of thesample or the migration thereof into another well connected thereto canbe prevented and the position of the sample in the microwell can beadjusted.

BACKGROUND ART

With the recent development and progress in nanotechnology, it has beenapractice to handle cells, proteins, genes and so on at a level ofseveral individuals. As a result, it becomes necessary to inject andtreat microsamples into containers (wells) for reaction, analysis ordetection. To carry out a series of reactions, analyses, detections,etc. on microchips, use is sometimes made of a structure wherein aplural number of wells are connected to each other each via a pipe, agroove or a channel. In such a case, attention should be taken toprevent the migration of a sample into the adjacent well due to theinjection pressure, which brings about some difficulties not only inmanual operations but also in operations with the use of an automaticinjection device. It is also desired to adjust the position of a sampleinjected into a microwell or to transfer the sample into the next wellwhile adjusting the position.

It is an object of the present invention to provide a structure to beused in the above-described apparatus whereby, in the step of injectinga microsample into a well, the migration of the sample into another wellor overflow from the well can be certainly prevented. It is anotherobject of the present invention to provide a structure wherein theposition of an injected sample in a well can be adjusted or the samplecan be transferred into the next well under controlling. It is stillanother object of the present invention to provide a microsampletreatment apparatus wherein a sample can be injected and transferredunder automated control.

It is still another object of the present invention to provide anapparatus for detecting chemotaxis of cells or separating chemotacticcells with the application of the structure having the functions asdescribed above.

DISCLOSURE OF THE INVENTION

The present invention relates to a microsample treatment apparatushaving a structure wherein a plural number of wells are connected toeach other via a part having resistance to fluids and the wells are eachprovided with tubes for injecting/sucking a sample and, if necessary,tubes for relieving pressure changes at the injection/suction,characterized in that these tubes have a space in common at the top endsthereof in which a liquid can be held. The part having resistance tofluids may be selected from among one or more thin pipes, narrow gaps,thin grooves, filters, resin-filled columns and other structures throughwhich a fluid can be passed but which have resistance to fluids.

The present invention further relates to a microsample treatmentapparatus wherein the top end of a tube formed in a well is locatedupper than the top ends of the tubes formed in one or more wellsopposite thereto across the part having resistance to fluids. Themicrosample treatment apparatus according to the present invention mayhave, in one or both of wells connected to each other via a channel, awall formed orthogonal to the channel to thereby restrict the amount ofa liquid in the vicinity of the channel.

The present invention relates to a microsample treatment apparatus whichcomprises a unit part having a single unit selected from the microsampletreatment apparatuses as described above, an integration unit having aplural number of units of the same or different types or a plural numberof integration units, a pipette or pipettes for controlling the liquidlevel in the unit part, and a system for controlling the operation ofthe liquid level control pipette(s). Moreover, the present inventionrelates to an automated microsample treatment apparatus characterized inthat the liquid level control pipette(s) are controlled so as to suck adefinite amount of a liquid contained in the space held in common by aplural number of tubes at the top ends thereof in each of the units inthe unit part, thereby adjusting the position of the sample in well(s)or transferring the sample into the respective next well(s) followed by,if necessary, supplying the liquid in a compensatory amount to returnthe liquid face to the original level. If necessary, the microsampletreatment apparatus may be provided with a sample reservoir, a specimenreservoir, pipette(s) washing part and sample supply pipette(s) andspecimen supply pipette(s) which are movable over these parts andfurther have a system for controlling the operations of these pipettes.The materials of the pipettes are not restricted to glass but can beappropriately selected from among metals, plastics and the like.

The present invention involves in its scope an apparatus for detectingchemotaxis of cells or separating chemotactic cells characterized inthat a plural number of wells are connected to each other via a parthaving resistance to fluids, the wells are each provided with tubes forinjecting/sucking a sample and, if necessary, tubes for relievingpressure changes at the injection/suction, these tubes have a space incommon at the top ends thereof in which a liquid can be held, and thewells are closely adhered to a glass substrate in the side opposite tothe tube side.

The present invention further relates to an apparatus for detectingchemotaxis of cells or separating chemotactic cells as described abovecharacterized in that the top end of a tube formed in a well for holdingcells is located upper than the top ends of the tubes formed in one ormore wells opposite thereto across the channel having resistance tofluids.

In the present invention, it is preferable that the channel havingresistance to fluids is a bank and a narrow gap is formed between thebank and the glass substrate. In this case, a terrace may be formed inthe upper part of the bank in the channel to form a gap between theterrace and the glass substrate. Alternatively, barriers constitutingone or more grooves having a width fit for the diameter or deformabilityof cells may be formed in the upper part of the bank and, if necessary,a terrace may be further formed together with the bank to form a gap fitfor the diameter or deformability of cells between the terrace and theglass substrate too. A plural number of grooves in the direction towardthe opposite well in the channel may be connected to each other via oneor more grooves orthogonal thereto. It is also possible that the widthof a plural number of grooves in the direction toward the opposite wellin the channel is changed stepwise each time the grooves intersect oneor more grooves orthogonal thereto. Furthermore, a plural number ofgrooves in the direction toward the opposite well in the channel may beformed by mutually shifting the positions thereof each time the groovesintersect one or more grooves orthogonal thereto. Moreover, arrays ofthe barriers constituting the grooves may be formed at two positions inboth sides of the terrace formed at the center of the bank. It is alsopossible that multistage terraces are formed on the bank in the channelso as to form gaps with different depths between the terrace and theglass substrate. In one or both of wells connected to each other via achannel, moreover, a wall may be formed orthogonal to the channel tothereby restrict the amount of a liquid in the vicinity of the channel.

The present invention relates to an automated apparatus for detectingchemotaxis of cells or separating chemotactic cells comprising a unitpart having a single unit selected from the apparatuses for detectingchemotaxis of cells or separating chemotactic cells as described above,an integration unit having a plural number of units of the same ordifferent types or a plural number of integration units, a cellreservoir, a specimen reservoir and liquid level control pipette(s),cell supply pipette(s) and specimen supply pipette(s) which are movableover these parts, and further having a detection part for detecting cellmigration in the unit part and, if necessary, recording the detectiondata which is integrated with the unit part or formed so as tocorrespond to a plural number of unit parts, and further having a systemfor controlling the movements of the liquid level control pipette(s),the cell supply pipette(s) and the specimen supply pipette(s) and, ifnecessary, a system for moving the unit part to the detection part andthe next unit part to the pipette flow line. If necessary, thisapparatus may further have a pipette washing part.

The present invention further relates to an automated apparatus fordetecting chemotaxis of cells or separating chemotactic cellscharacterized in that the operations of the respective pipettes arecontrolled as follows: after optionally stirring, a definite amount acell suspension is sucked by the cell supply pipette(s) and suppliedinto the unit part; then a definite amount of a liquid, which iscontained in the space held by the top ends of a plural number of tubesin common in each unit, is sucked by the liquid level control pipette(s)to thereby adjust the position of the cells in the wells; the liquid inthe compensatory amount is supplied from the liquid level controlpipette(s) into the space to thereby return the liquid face to theoriginal level; then a definite amount of a specimen is sucked from thespecimen reservoir by the specimen supply pipette(s) and supplied intothe unit part; then the pipettes move toward the pipette washing part inwhich they are washed by repeatedly sucking and discharging the washingliquor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a model view which shows an example of an apparatus fordetecting chemotaxis of cells or separating chemotactic cells previouslyproposed by the present inventors.

FIG. 2 is a bottom plan view of the apparatus of FIG. 1.

FIG. 3 is a model view which shows an example of the application of thestructure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells. The arrowshows the liquid level of a liquid filling up the apparatus.

FIG. 4 is a model view showing another example of the application of thestructure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells which isprovided with tubes 3 for injecting/collecting a sample into wells andtubes 4 for relieving decrease/increase in pressure at the step ofinjecting/collecting the sample. The arrow shows the liquid level of aliquid filling up the apparatus.

FIG. 5 is a model view showing another example of the application of thestructure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells whereinthe top end 3Ab of a tube 3A in a well 2A for holding cells is locatedupper than the top end 3Bb of a tube 3B in another well 2B. The arrows Iand II show the liquid levels of a liquid filling up the apparatus.

FIG. 6 is a model view showing another example of the application of thestructure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells which isprovided with tubes 3 for injecting/collecting a sample into wells andtubes 4 for relieving decrease/increase in pressure at the step ofinjecting/collecting the sample, wherein the top ends 3Ab and 4Ab oftubes 3A and 4A in a well 2A for holding cells are located upper thanthe top ends 3Bb and 4Bb of tubes 3B and 4B in another well 2B. Thearrows I and II show the liquid levels of a liquid filling up theapparatus.

FIG. 7 shows a modification example of the structure as shown by FIG. 6.The arrows I and II show the liquid levels of a liquid filling up theapparatus.

FIG. 8 is a top plan view of a substrate in an example wherein wells areconnected each via a channel in the triple system.

FIG. 9 is a top plan view of a substrate in an example wherein a pluralnumber of wells 2B₁₋₄ are connected to a single well 2A each via achannel 1.

FIG. 10 is a sectional view of an apparatus having the substrate asshown by FIG. 9 along the dashed dotted line in FIG. 9. The arrows I andII show the liquid levels of a liquid filling up the apparatus.

FIG. 11 is a top plan view of an example wherein the connection systemin FIG. 9 is provided circularly.

FIG. 12 shows an example of the structure of a channel 1.

FIG. 13 shows an example of the arrangement of barriers 12 and grooves13 in a channel 1. The arrow shows the direction toward the oppositewell.

FIG. 14 is a sectional view of the channel 1 shown by FIG. 13.

FIG. 15 shows an example wherein grooves 13 in the direction toward theopposite well across a channel 1 are connected via two grooves 14orthogonal thereto. The arrow shows the direction toward the oppositewell.

FIG. 16 shows an example of an integration of multiplicity of unitswherein the units are all in the same type.

FIG. 17 shows an example of an integration of multiplicity of unitswherein the units are in different types.

FIG. 18 shows an example wherein multiplicity of units are circularlyintegrated.

FIG. 19 is a sectional view along the dashed dotted line in FIG. 18.

FIG. 20 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells or separating chemotactic cells wherein(1) is perspective views of individual parts and (2) is sectional viewscorresponding thereto.

FIG. 21 is a model view of an apparatus wherein a well to be reacted andanother well for holding the target substance are connected via acolumn. The arrows I and II show the liquid levels of a liquid fillingup the apparatus.

FIG. 22 is a model view of an apparatus for separating substances. Thearrows I and II show the liquid levels of a liquid filling up theapparatus.

FIG. 23 shows an example wherein a bank 8 in a channel 1 has multistageterraces 11 ⁻¹⁻⁴.

FIG. 24 shows an example of wells wherein walls are formed along achannel.

FIG. 25 shows another example of wells wherein walls are formed along achannel.

FIG. 26 shows an arrangement example wherein the wells shown by FIG. 24are integrated.

FIG. 27 shows an example of an automatic controlling system of theapparatus according to the present invention.

FIG. 28 shows the movement of liquid level control pipette(s).

FIG. 29 shows an example of containers in a cell reservoir.

FIG. 30 shows an example of a container in a specimen reservoir.

FIG. 31 shows an arrangement example of the containers shown by FIG. 30in the specimen reservoir.

FIG. 32 shows another example of a container in the specimen reservoir.

FIG. 33 shows an arrangement example of the containers shown by FIG. 32in the specimen reservoir.

FIG. 34 shows an example of a pipette to be used in the presentinvention.

FIG. 35 shows an example wherein pipette tip inlets are formed in theupper part of tubes for injecting/collecting a sample.

FIG. 36 shows an example wherein grooves in the direction toward theopposite well across a channel are connected to each other via twogrooves formed orthogonally thereto and the width of the grooves in thedirection toward the opposite well is changed stepwise each time thegrooves intersect the grooves orthogonal thereto. Each arrow shows thedirection toward the opposite well. In this figure, the width of thebarriers per se is changed.

FIG. 37 shows an example of the modification of the structure of FIG. 36in which the barriers have the same size but are changed in number. Thearrow shows the direction toward the opposite well.

FIG. 38 shows an example wherein grooves in the direction toward theopposite well across a channel are connected to each other via threegrooves formed orthogonally thereto and the grooves in the directiontoward the opposite well are formed by mutually shifting the positionsthereof each time the grooves intersect the grooves orthogonal thereto.In this figure, the grooves shift by ½ pitch toward the orthogonaldirection. Each arrow shows the direction toward the opposite well.

FIG. 39 shows an example wherein barriers are jointed in the directiontoward the opposite well. Each arrow shows the direction toward theopposite well.

FIG. 40 shows an example wherein a terrace is formed at the center of abank and two arrays of barriers are formed in both sides of the terrace.

DESCRIPTION OF THE REFERENCE NUMERALS AND SIGNS

-   -   1: channel.    -   2: well. Appendixes A, B, B_(1-n), and C are provided to        differentiate the wells.    -   3: tube for injecting/collecting samples. Appendixes A, B,        B_(1-n), and C are provided to differentiate the wells. Appendix        a represents a penetrating hole corresponding to a tube 3 of a        substrate 5. Appendix b represents the top end of the tube 3.    -   4: tube for avoiding increase/decrease in pressure at        injecting/collecting samples. Appendixes A, B, B_(1-n), and C        are provided to differentiate the wells. Appendix a represents a        penetrating hole corresponding to a tube 4 of a substrate 5.        Appendix b represents the top end of the tube 4.    -   5: substrate.    -   5′: packing.    -   6: glass substrate.    -   7: block having tube mounted thereto.    -   8: bank.    -   9: detector.    -   10: space held together by top ends of tubes.    -   11, 11 _(−1 to 4): terraces.    -   12: barrier in channel 1.    -   13: groove in the direction toward the opposite well across        channel.    -   14: groove formed orthogonally to groove 13.    -   15: magnet.    -   16: column located between wells.    -   17: cover cap.    -   18: O-ring.    -   19: guide pin receiver hole.    -   20: guide pin.    -   21: intermediate base.    -   22: bottom base.    -   23: bottom substrate.    -   24: wall formed along channel.    -   25: cell reservoir.    -   26: cell injection part.    -   27: liquid injection part.    -   28: specimen reservoir.    -   29: pipette tip inlet port.    -   30: pipette washing part.    -   31: multichannel syringe.    -   32: actuator.    -   33: needle of automatic pipette.    -   34: tip of manual pipette.    -   ←: level of liquid filling up apparatus.    -   ←I: level of liquid making the top end of upper tube submerged.    -   ←II: level of liquid making the top end of upper tube visible        above the liquid face.    -   X-X′: flow line of specimen supply pipette.    -   Y-Y′: flow line of cell supply pipette.    -   Z-Z′: flow line of liquid level control pipette.

BEST MODE FOR CARRYING OUT THE INVENTION

The microsample treatment apparatus according to the present inventionprovided with wells into which a sample such as a liquid or a suspensionis injected is an apparatus for handling organic or inorganic chemicals,polymers such as proteins, genes, cells and so on in the state ofsolutions or suspensions. Although the structure of the presentinvention is not specifically restricted in the amount of samples to betreated, it is expected that high technical merits can be achievedthereby in case of using samples of the order of several milliliters tomicroliters.

The present invention is applied to case wherein a plural number ofwells are connected to each other via a structure having resistance tofluids and the wells are each provided with tubes for injecting orsucking a sample and, if necessary, tubes for relieving pressure changesat the step of the injecting or sucking the sample. That is, such anapparatus has a plural number of tubes as a whole. In the presentinvention, these tubes have a space in common at the top ends thereof inwhich a liquid can be held. Owing to this structure, unexpectedmigration and overflow caused by a rapid change in pressure in the wellsin the step of injecting or sucking a sample or unexpected migration ofthe sample caused by horizontal off balance of the apparatus can beeffectively prevented.

By employing the structure wherein a plural number of tubes have a spacein common at the top ends thereof in which a liquid can be held, theposition of a sample can be adjusted in a microwell or the sample can betransferred into the next well under controlling, in case of handlingsamples the position of which should be adjusted in the well or whichshould be transferred into the next well. To further ensure the controland migration, the top end of a tube formed in the well for holding thesample is located upper than the top ends of tubes formed in otherwells.

To enable the migration of a sample among a plural number of wells, thewells are usually connected to each other via, for example, thin pipes,narrow gaps, thin grooves, filers, resin-filled columns or channels. Thepresent invention relates to an apparatus wherein a plural number ofwells are connected to each other via such a structure having resistanceto a fluid flow.

Now, illustration will be made on the application the present inventionto an apparatus wherein a plural number of wells are connected to eachother each via a channel, for example, an apparatus for detectingchemotaxis of cells or separating chemotactic cells. However, it isobvious from the description given above that the present invention isnot restricted to apparatuses for detecting chemotaxis of cells orseparating chemotactic cells but applicable to various apparatuses.

In the apparatus for detecting chemotaxis of cells or separatingchemotactic cells, a cell suspension is put into one of the wells whilea specimen solution is put into the other well. Then it is detectedwhether or not cells migrate toward the well holding the specimensolution, or cells which have migrated are selectively collected. Inthis apparatus, for example, the well holding the cell suspension isconnected to the well holding the specimen solution via a channel. Thus,the state where the cells are passing through the channel is observed,or the cells which are passing or have passed through the channel arecounted.

A channel which makes it possible to observe or detect the passage ofindividual cells has resistance to fluids. In an apparatus provided withsuch channels, it is sufficient to employ only a small amount of cellsas a sample, which brings about a merit of being adequate for examiningrare cells. In addition, there is another merit that quantitativeanalysis can be made. In this case, however, the whole apparatus is in asmall size and thus samples should be handled in microquantities. As aresult, there frequently arises unexpected migration of cells toward awell holding a specimen solution under the effect of an increase inpressure caused by the injection into the wells. In case the wells arenot held horizontally after the injection, moreover, cells wouldmigrate. These unexpected migrations of cells result in confusion in thejudgment whether the specimen is a chemotactic factor or not. Toaccurately detect the migration of cells toward a well holding aspecimen solution by their own actions, it is therefore required toprevent the migration of the cells at the point of injecting a sample orafter the injection.

As one of countermeasures thereto, the present inventors have proposed astructure wherein each well has a tube for injecting a sample and anadditional well connected thereto for relieving an increase in pressureat the injection (Japanese Patent Application No. 2001-226466). Now,this structure will be briefly described by reference to FIGS. 1 and 2.

In the apparatus shown by FIG. 1, a cell suspension is injected into awell 2A through a tube 3A. A specimen solution is injected into a well2B through a tube 3B. In case where this specimen contains a chemotacticfactor, cells tend to migrate from the well 2A toward the well 2B andthus pass through the channel 1. In FIG. 1, a gap corresponding to thecell size is provided between a bank 8 formed on a substrate 5 and atransparent glass substrate 6. Alternatively, barriers constituting aplural number of thin grooves through which individual cells can passmay be formed. The state of the cells passing through the channel 1 canbe observed by, for example, a microscope 9 through the glass substrate6. FIG. 2 is a bottom plan view of the substrate 5.

In the apparatus shown by FIGS. 1 and 2, the tubes 3A and 4A and thetubes 3B and 4B are connected to each other in the respective wells. Inthis structure, pressure is dispersed through the tubes connected toeach other. In the present invention, in contrast thereto, the top endsof all of the tubes formed in respective wells have a space in common inwhich a liquid can be held. Owing to this structure, the migration atthe injection can be more surely relieved or the migration can becontrolled (see FIGS. 3 and 4).

FIG. 3 shows an example of the structure according to the presentinvention which is a unit consisting of a substrate 5, a block 7 and aglass substrate 6. In FIG. 3, a space 10 is held in common by the topends 3Ab and 3Bb of tubes 3A and 3B formed in respective wells. Thewhole apparatus is filled up with a liquid not affecting chemotaxis suchas a buffer solution. The amount of the liquid is sufficient for atleast filling up a part of the space 10. Owing to this liquid, the wholeapparatus is maintained under a definite pressure. Moreover, theresistance of the liquid contributes to the prevention of rapidmigration of a sample caused by the injection pressure and horizontaloff balance of the wells. FIG. 4 shows another example of the structureaccording to the present invention. In this unit, wells are providedwith tubes 3A and 3B for injecting a sample and, further, tubes 4A and4B connected thereto and a space 10 is provided by the top ends 3Ab,4Ab, 3Bb and 4Bb of all of these tubes in common.

In the step of collecting the migrated cells by sucking from a wellholding the specimen through a tube formed in the well, the innerpressure is reduced and thus the samples in wells are mixed each other.In the structure as shown by FIG. 4, this phenomenon can be particularlyeffectively relieved.

In case of detecting chemotaxis of cells or separating cells, it ispreferable that the injected cells are first brought together in thevicinity of a channel in a well. In case of the apparatus for detectingchemotaxis of cells or separating chemotactic cells as shown by FIG. 3,for example, it is preferable that cells injected into the well 2Athrough the tube 3A are located in the vicinity of the channel 1.Namely, these cells may be considered as an example of a sample whoseposition in a well should be adjusted. This position adjustment can becarried out by sucking an appropriate amount of the liquid at anappropriate speed from the well 2B located oppositely across the channelthrough the tube 3B. The amount of the liquid to be sucked is determinedbased on the capacities of the tube and the well after discharging theliquid from the space 10. The amount of the liquid to be sucked and thesucking speed can be easily controlled by a computerized program.

The present invention further involves in its scope, as a modificationof the above-described structure, a microsample treatment apparatus suchas an apparatus for detecting chemotaxis of cells having a structurewherein the top end of a tube formed in a well for holding, for example,a cell suspension is located upper than the top end of a tube formed inanother well opposite thereto across a channel (see FIGS. 5 to 7). InFIG. 5, a block 7 having a tube mounted thereon has been cut downwardaround the top end 3Bb of a tube 3B formed in a well 2B. Thus, the topend 3Ab of the tube 3A in a well 2A is located upper than the top end3Bb of the tube 3B. At first, the amount of a liquid filling up thewhole apparatus is controlled so that the liquid level is located abovethe top end 3Ab of the tube 3A, i.e., the position indicated by thearrow I in the figure. When cells are injected into the well 2A throughthe tube 3A in this state, rapid migration of the cell is prevented dueto the uniform pressure in the whole apparatus and the resistance of theliquid. Thus, the cells scatter in the tube 3A and the well 2A. Next,the liquid is sucked off from the space 10 so that the liquid level islowered to the position indicated by the arrow II (i.e., such a level asmaking the tope end 3Ab of the tube 3A visible above the liquid face3A).Further, an appropriate amount of the liquid is sucked off and thus thecells scattering in the vicinity of the channel in the well 2A can bebrought together. The amount of the liquid to be sucked off can becalculated based on the capacities of the tube 3A and the well 2A. Inusual, the object can be achieved by sucking off the liquid in an amount1/10 to 1/3 times as much as the capacities. By injecting the specimensolution into the well 2B after returning the liquid level to theposition indicated by the arrow I, a rapid change in pressure at theinjection can be relieved.

As the liquid employed for returning the liquid level to the positionindicated by the arrow I, it is preferable to use a liquid having alower specific gravity than the liquid preliminarily contained in theapparatus (e.g., an aqueous solution such as a buffer solution). Thus,the upper part of the tubes in each well can be covered with the liquidhaving the lower specific gravity and thus the unnecessary diffusion ofthe sample can be prevented owing to the covering effect. An arbitraryliquid can be selected therefor so long as it is inert to the sample,insoluble in water and has a specific gravity lower than 1.0. Examplesthereof include Mineral Water M3516 (specific gravity: 0.84,manufactured by Sigma) and liquid paraffin.

FIG. 6 shows an example of a unit having tubes 3A and 3B for injecting asample and tubes 4A and 4B connected thereto in each well, wherein thetop ends 3Ab and 4Ab of the tubes in a well 2A are located upper thanthe top ends 3Bb and 4Bb of the tubes in another well 2B. FIG. 7 showsan example wherein a slope is formed on a block 7 so that the top endsof tubes in a well 2A are located upper. These figures show the exampleswherein the top ends of some tubes are located upper than the top endsof other tubes. Various modifications can be further made to achieve thesame object.

The above-described structure wherein the top ends of some tubes arelocated upper than the top ends of other tubes is effective in theconnecting manners as will be described hereinbelow. If necessary, otherunit(s) may be further jointed and connected to a double system forconnecting wells each via a channel as shown by FIGS. 3 to 7 to therebygive, for example, a triple system shown by FIG. 8. In FIG. 8, forexample, cells are put into a well 2A, a chemotactic factor is put intoa well 2C and a specimen solution is put into a well 2B. Thus, it can beexamined whether or not the specimen solution inhibits the chemotacticfactor. Moreover, multiple systems are applicable to various purposes.

As FIG. 9 shows, it is also possible to construct a so-called concentricsystem wherein a plural number of wells are connected to each other eachvia a channel around a single well. Furthermore, a concentric circularsystem as shown by FIG. 11 may be constructed as a modification of thetype of FIG. 9. Although a triple system is employed in the example ofFIG. 11, it is also possible to employ a double system. In the exampleof FIG. 9, a tube 3A is mounted to a penetrating hole 3Aa. Similarly,tubes 3B₁₋₄ are mounted to penetrating holes 3B_(1a-4a) while tubes4B₁₋₄ are mounted to penetrating holes 4B_(1a-4a) respectively. A cellsuspension is supplied into a well 2A through the tube 3A and variousspecimens are supplied into wells 2B₁₋₄. Thus, a plural number ofchemotactic factors can be examined at the same time. By supplying asample containing a plural types of cells, the cells can be separateddepending on types at once (i.e., sorting). For example, chemotacticfactors corresponding to respective cell types are put into the wells2B₁₋₄ and a sample containing plural types of cells (for example, wholeblood) is supplied into the central well 2A. Then the cells contained inthe sample migrate toward the wells 2B₁₋₄ containing the correspondingchemotactic factors. After a definite period of time, the cells arecollected from each of the wells 2B₁₋₄ through the tubes 3B₁₋₄ or cellshaving migrated into the wells 2B₁₋₄ are identified.

In the well-connecting manners as shown by FIGS. 8, 9 and 11, the tubes3 and 4 are connected to each other in the well 2 in which they areprovided. In these connecting manners, all of the tubes hold at the topends thereof a space 10 in common. The top ends of the tubes in a wellinto which cells are injected are located upper than the top ends ofother tubes. Then a liquid is supplied so that the top ends of the tubesin the well into which cells are injected are submerged (see FIG. 10).FIG. 10 is a sectional view of the apparatus shown by FIG. 9 along thedashed and dotted line. In this example, the top ends 3Ab and 4Ab oftubes 3A and 4A in a well 2A are located upper than the top ends3B_(1b-4b) of tubes 3B₁₋₄ in other wells 2B₁₋₄. An arrow I shows thatthe level of the liquid filling up the space 10 is located above the topends 3Ab and 4Ab of the tubes 3A and 4A. Cells injected into the well 2Athrough the tube 3A scatter in the tube 3A and the well 2A. Then theliquid in the space 10 is sucked off and thus the liquid level islowered to the position indicated by another arrow II so that the topend 3Ab of the tube 3A becomes visible above the liquid face. Then anappropriate amount of the liquid is further sucked off. Thus, the cellsin the well 2A can migrate toward the wells 2B₁₋₄ and thus are broughttogether in the vicinity of the channel 1 toward respective wells. Theamount of the liquid to be sucked off can be calculated based on thecapacities of the tube 3A and the well 2A. Thus, the chemotaxis of thecells in the well 2A concerning the wells 2B₁₋₄ can be examined underthe same positional conditions.

As another example of the embodiment to which the structure of thepresent invention can be applied, an apparatus shown by FIG. 21 may becited. Namely, FIG. 21 is a model view of an apparatus wherein areaction is carried out in a well 2A followed by a treatment through acolumn 16 and then unadsorbed substances passing through the column arecollected from a well 2B. In this case, the column serves as an obstaclehaving resistance to fluids. Substances to be reacted are put into thewell 2A in the state that the liquid level is at the position indicatedby an arrow I. After the completion of the reaction, the liquid level islowered to the position indicated by another arrow II. After furthersucking, the reaction mixture migrates from the well 2A toward thecolumn 16. By further sucking, a substance passing through the columnmigrates toward the well 2B. In case where the substance adsorbed by thecolumn is the target substance, the eluate is supplied into the columnvia the well 2A. Thus, the eluted substance can be collected in the well2B.

In addition to the above-described case, various applications can bemade. That is to say, interactions among substances can be examined atthe level of microquantities by controlling the migration of samplesamong wells which are connected to each other. For example, theseapparatuses are applicable to antigen/antibody reactions,enzyme/substrate reactions, reactions between soluble receptors andligands, and so on.

In the well 2A of the apparatus shown by FIG. 5, for example, anantibody bonded to plastic beads of a definite size is reacted with anantigen protein. Then the liquid level is lowered from I to II andfurther the liquid is sucked off. Thus the unreacted antigen proteinpasses through the groove in the barrier 12 and migrates into the well2B. However, an antigen which has reacted with the antibody bonded tothe plastic beads cannot pass through the groove in the barrier 12because of the presence of the beads, thereby being separated from theunreacted antigen protein. Thus, substances can be separated byappropriately selecting the combination of the bead size and the groovewidth.

It is also possible to use magnetic beads. For example, magnetic beadshaving a uniform particle size, which are composed of polymer coreshaving a magnetizable substance (for example, γFe₂O₃, Fe₃O₄) uniformlydistributed therein and a hydrophilic polymer coating, are commerciallyavailable (Dynabeads® manufactured by DYNAL, Norway). By bonding variousantibodies onto the surface of these beads, the magnetic beads can bebonded to cells or proteins. By bringing close to a powerful magnet(MPC), the magnetic beads are magnetized and attracted to the magnet.When the magnet is moved away, the beads are demagnetized and thusscatter again. These characteristics have been used in purifying cells,proteins, etc. For example, Kanegasaki, S. et al. isolated peripheral Blymphocytes by using magnetic polystyrene beads (manufactured by DYNAL)coated with CD19 antibody (J. Biochem., 117:758-765 (1995)).

In an apparatus shown by FIG. 22, a protein mixture and antibodieslabeled with magnetic beads (magnetic antibody beads) are injected intoa well 2A at the liquid level I. After adsorbing a protein reacted withthe antibody by a magnet 15 provided at the bottom of the well 2A, theliquid level is lowered to II. Then the liquid is sucked off from aspace 10. Thus, a protein unadsorbed by the magnet 15 alone migratesinto another well 2B. By selecting an appropriate antibody, a desiredprotein can be thus separated or an unnecessary protein can be thuseliminated. For separate proteins with the use of magnetic materials, ithas been a practice to use columns. However, treatments with columns canbe performed on the milliliter scale and, therefore, are unsuitable fortreating proteins in microquantities. By using the apparatus accordingto the present invention, proteins can be separated even on the scale ofseveral microliters or less.

The present invention makes it possible to downsize the whole apparatusand thus samples can be treated in microquantities. Moreover, it ispossible to integrate multiplicity of units and thus a large number ofspecimens can be treated at the same time. In addition, the treatmentcan be easily automated by programmed control of suction and injectionof liquids.

That is to say, the apparatus can be automated by providing a unit parthaving a single unit, an integration unit having a plural number ofunits of the same or different types or a plural number of integrationunits, liquid level control pipette(s) and a system for controlling themovements of the liquid level control pipette(s). The operations of theliquid level control pipette(s) are controlled as follows. Namely, adefinite amount of a liquid, which is contained in the space held by thetop ends of a plural number of tubes in common in each unit, is suckedby the liquid level control pipette(s) to thereby adjust the position ofa sample in the well, or transfer the sample into the next well and, ifnecessary, the liquid in the compensatory amount is supplied from theliquid level control pipette(s) into the space to thereby return theliquid face to the original level. These controlling operations can beeasily carried out by computerized programming.

It is also possible to automate the whole apparatus involving the stepsof supplying and collecting a sample, a specimen, a reagent, etc. byproviding a unit part, a sample reservoir, a specimen reservoir andsample supply pipette(s) and specimen supply pipette(s) movable overthese parts and further a system for controlling the operations of thesepipettes. If necessary, it is also possible to add a pipette washingpart and a system for controlling the operation of washing the pipettesin the pipette washing part.

Next, the structure of the apparatus according to the present inventionwill be described in greater detail by reference to an apparatus fordetecting chemotaxis of cells as an example. However, it is to beunderstood that the present invention is not restricted to an apparatusfor detecting chemotaxis of cells but applicable to other apparatuses inorder to solve similar technical problems as discussed above.

1) Structure of Unit

As FIG. 3 shows, a channel 1 and wells 2A and 2B are integrally formedon a substrate 5. The substrate 5 has holes (penetrating holes) 3Aa and3Ba for mounting tubes 3A and 3B connected to respective wells. A block7 having the tubes 3A and 3B is fixed so as to fit for the penetratingholes 3Aa and 3Ba. In the upper part of the block, a space 10 commonlyheld by the top ends 3Ab and 3Bb of the tubes 3Aand 3B is provided. Thebottom face of the substrate 5 is adhered to an optically polished glasssubstrate 6. The block 7, the substrate 5 and the glass substrate 6 maybe pressed and fixed by fastening, for example, with an O-ring or apacking (see FIG. 20). Alternatively, the substrate 5 and the glasssubstrate 6 may be integrally formed. Alternatively, the substrate 5,the glass substrate 6 and the block 7 may be integrally formed. As FIG.4 shows, the tubes formed in the wells 2A and 2B may be further providedwith tubes 3A, 3B, etc. for injecting/collecting a sample and tubes 4A,4B, etc. for relieving pressure changes. As FIGS. 5, 6. etc. show, thespace 10 may be partly cut downward to form a concave. Alternatively, aslope may be formed as shown by FIG. 7, etc.

2) Well

Wells 2 are formed for holding a sample (i.e., a cell suspension) or aspecimen solution such as a solution containing a chemotactic factor ora solution containing an inhibitor therefor. The capacity of the wellsis not particularly restricted, so long as a liquid can be held thereinin the minimum amount needed. For example, it is sufficient that thedepth ranges from about 0.05 to about 0.1 mm, the width is about 1.2 mmand the length is about 2.5 mm. It is also possible to provide a wallorthogonal to a channel in one or both of wells connected to each othervia the channel (for example, the well for holding cells) to therebyrestrict the amount of the liquid in the vicinity of the channel. Thus,the position of cells in the well can be adjusted (FIG. 24). FIG. 24shows an example wherein wells 2A and 2B are connected to each other viaa channel 1 and walls 24A and 24B are formed in respective wellsorthogonally to the channel 1. Although the distance between the walls24 and the channel 1 may be arbitrarily determined, it usually rangesfrom 50 to 300 μm.

FIG. 25 shows modification examples of the well unit having wallsprovided orthogonally to the channel. That is, FIG. 25(1) shows anexample wherein a channel is formed in a part of the well width; (2)shows an example wherein a channel is halved at the center, a couple ofwells (2B, 2C) are provided opposite to a single well (2A) across thechannel, and a wall 24 is formed exclusively in the well 2A side; and(3) shows an example wherein two arrays of barriers are formed in bothsides of a terrace 11 in a channel. Needless to say, these modificationsare cited merely by way of example and thus the present invention is notrestricted thereto. If necessary, a terrace may be formed between thewall provided orthogonally to the channel and the bank.

3) Channel

Now, an example of the structure of a channel 1 (FIGS. 1, 3 and 4) willbe illustrated by reference to FIG. 12. The channel 1 is a spaceprovided between a bank 8 (a convex on a substrate 5) partitioning wells2A and 2B at both ends and a glass substrate 6. The bank 8 partitioningthe wells 2A and 2B formed at both ends of the channel 1 is notrestricted in size. For example, the height of the bank 8 may range fromabout 0.03 to about 0.1 mm, while the length in the direction toward theopposite well may range from about 0.01 to about 0.5 mm and the lengthin the direction orthogonal to the direction toward the opposite wellmay be about 1.2 mm.

In a preferred embodiment, a plural number of barriers 12 are formed onthe bank to thereby constitute grooves 13 through which cell pass, asshown by FIGS. 13 to 15. In case where no barrier constituting groovesis formed in the upper part of the bank, a terrace providing a gap or adepth fit for the diameter or deformability of cells is formed betweenthe upper face of the bank and the glass substrate. In this case, thedepth usually ranges from 3 to 50 μm depending on the type of cells.That is to say, the width may range from 3 to 10 μm (for example, 4, 5,8 or 10 μm) in case of neutrophils, eosinophils, basophils,monocytes/macrophages, T cells, B cells and the like, and from 8 to 20μm in case of cancer cells and cells existing in tissues.

By forming flat terraces in both sides of the barriers on the upper faceof the bank, the passage of cells can be more easily observed. Thus, itis favorable to form terraces 11 (FIG. 12), though they are notessentially required. In case of providing the terraces 11, the lengththereof in the direction toward the opposite well appropriately rangesfrom about 0.01 mm to about 0.5 mm.

By forming multistage terraces 11 as FIG. 23 shows, cells put into wellsin one side can be easily brought together in the vicinity of the bank 8by sucking from the other side to adjust the position of the cells inthe well. In case where the cells are neutrophils, eosinophils,basophils, etc., for example, the distance between the terraces 11 ⁻²and 11 ⁻³ and a glass substrate 6 (i.e., corresponding to the height ofa barrier 12 in the figure) is set to 3 μm and the distance between theterraces 11 ⁻¹ and 11 ⁻⁴ and a glass substrate 6 is set to 4.5 μm. Thencells are supplied into a well 2A and the liquid is sucked from the sideof another well 2B. Then the cells once stop at the terrace 11 ⁻¹. Next,the cells are liable to bring together between the terrace 11 ⁻² and theglass substrate 6. The distance between each of the terraces 11_(−1 to 4) and the glass substrate 6 can be arbitrarily determineddepending on the sample to be treated. Although these distances usuallyrange from about 3 to 5 μm, the present invention is not restrictedthereto. When the terrace (11 ⁻³) in the side opposite to the wellscontaining the cells is made about 1.5 to 5 times longer than theterrace (11 ⁻²) in the side of the wells containing the cells, the cellshaving passed through the channel can be more easily observed andcounted. Although a barrier 12 is formed in the example shown by FIG.23, the barrier is not always necessary in case where the distancebetween the terraces 11 ⁻² and 11 ⁻³ and the glass substrate 6corresponds to the diameter or deformability of cells.

In case where barriers 12 (see FIGS. 12 to 14) are formed on the upperface of the bank, grooves 13 constituted by the barriers 12 may have anarbitrary cross-sectional shape, for example, a V-shaped section, aconvex section or a semicircular section. It is preferable that thegrooves 13 have a width fit for the diameter or deformability of cells.The term “deformability” of cells as used herein means that, in case offlexible cells, the cells can easily change their shape (for example,into flat or string-shaped cells) owing to the flexibility and thus canpass through a gap having a smaller size than the diameter of the cellsbeing in the inherent spherical shape in a free space. By forming suchgrooves, cells can be observed at individual level and thus separateddepending on desired types. The width of a groove 13 usually may rangefrom 3 to 50 μm. It is preferable that the width allows the passage ofcells one by one. Thus an appropriate width may be selected depending onthe cell type. The width may range from 3 to 10 μm (for example, 3, 5, 8or 10 μm) in case of neutrophils, eosinophils, basophils,monocytes/macrophages, T cells, B cells and the like, and from 8 to 20μm in case of cancer cells and cells existing in tissues. The number ofthe grooves 13 is determined depending on the width of the barriersconcerning the channel width and the groove width. In case where thechannel width is 1 mm, the barrier width is 10 μm and the groove widthis 5 μm, for example, the number of grooves is 66 at the largest. Tosmoothly perform the detection and observation, the number of thegrooves 13 preferably ranges from 1 to about 100, still preferably fromabout 10 to about 70.

The length of the barriers 12 ranges from about 5 to about 400 μm. Forexample, use may be made of a barrier length of 5, 15, 20, 30, 40, 60,100, 200, 300 or 400 μm. The width of the barriers 12 per se can beappropriately determined. In case of employing the structure as will beshown in FIG. 38 hereinafter, it is effective that the width and lengthof the barriers are almost the same.

As FIG. 15 shows, the grooves 13 constituting the channel 1 may beconnected to each other via one or more grooves 14 orthogonal to thedirection toward the opposite well. Owing to this structure, thediffusion of a substance put into one well toward the other well can beuniformized, or cells under passage can be more accurately understood.In this case, the width of the grooves 13 may be changed stepwise eachtime the grooves intersect grooves 14 orthogonal thereto in thedirection toward the opposite well (see FIGS. 36 and 37). Alternatively,grooves in the direction toward the opposite well may be formed bymutually shifting the positions thereof each time the grooves intersectgrooves orthogonal thereto (see FIG. 38). FIG. 38 shows an examplewherein the grooves are formed as shifting by ½ pitch in the orthogonaldirection. It is also possible that the barriers are jointed to eachother in the direction toward the opposite well (see FIG. 39).Alternatively, arrays of barriers can be formed in two positions in bothside of a terrace which is formed at the center of the bank (see FIGS.25(3) and 40). By using these structures, cells having passed thegrooves can be easily observed and counted. It is desirable that theterrace located at the center has an area which can be included in themicroscopic field. FIG. 40(1) is a top plan view while (2) is asectional view.

The height of the barrier 12 (i.e., the depth of the grooves) may beappropriately determined depending on the depth of focus of theobjective lens of a microscope, a CCD camera, etc. to be used inobserving the cell migration. For example, a depth of about 3 to about4.5 μm is preferable in case of an objective lens having a focus depthof 10 to 40× magnification, though the present invention is notrestricted thereto.

4) Construction of Well and Channel

As a material of the substrate 5, it is preferable to use single-crystalsilicon which can be easily fine processed and is relatively inert tocells. The barriers 12 and the grooves 13 in the channel 1 can beconstructed by subjecting the single-crystal silicon to photolithographyor etching (for example, wet etching or dry etching) employed inmanufacturing integrated circuits. The wells 2 and the penetrating holes3 a and 4 a, which are larger than the barriers 12 and the grooves 13,can be constructed by using various known engineering techniques such assand blasting and dry etching. In addition to single-crystal silicon,use can be made of hard glasses, hard plastics, metals, etc., so long asa microstructure can be constructed in the channel. In case of usingplastics, it is preferable to employ a treatment for making the surfacehydrophilic, for example, forming a hydrophilic film on the surface. Itis also possible to separately construct the channel 1 and the wells 2and then combine them together.

5) Block and Tube

As shown by FIG. 3, the block 7 is a member located on the substrate 5and having tubes connected to wells. The tubes usually have a square orcircular cross-sectional shape. Although these tubes are not restrictedin size, a square tube has a side length of about 1 mm while a roundtube has a diameter of about 1 mm in usual. To hold a cell suspension ora specimen solution in a desired volume, it is necessary that thesetubes have a length of about 2 to about 10 mm. The materials of theblock or tubes maybe selected from among glasses, plastics such asacrylic resins and metals. The tubes can be easily produced by usingcommonly employed engineering techniques such as mechanical drilling orlaser drilling. Similarly, the space held commonly by the top ends ofthe tubes can be formed above the block 7 by usual engineeringtechniques.

To inject cells or a specimen into each unit by hands (i.e., manually),the periphery of the top end of each supply tube may be cut downward tothereby form a funnel-shaped concave. Thus, a pipette can be easilyinserted (29 in FIGS. 35(1) and (2)).

6) Glass Substrate

As shown by FIG. 3, the glass substrate 6 is tightly pressed on thesubstrate 5 to provide a space in which a liquid is held, therebyenabling the observation of cells passing through the channels. Thus,the glass substrate 6 should remain optically transparent and flat andprovide a plane to which cells can adhere. Use can be made therefor ofglass as well as plastics such as transparent acrylic resins, so long asthe above objects can be achieved thereby. Although its thickness is notparticularly restricted so long as no strain arises in the step ofpressing onto the substrate, the thickness adequately ranges from 0.7 to2 mm.

7) Arrangement of Multiplicity of Units

By referring a plural number of wells connected to each other each via achannel as a single unit, a plural number of units may be arranged andintegrated on a single substrate. Thus, an apparatus whereby a largenumber of specimens can be treated at the same time can be obtained.Units of the same type may be arranged in parallel or units of differenttypes may be arranged. Next, the types of the arrangement andintegration will be described by reference to respective figures.However, it is to be understood that the present invention is notconstrued as being restricted thereto and thus various combinations maybe also employed depending on the purpose.

FIG. 16 shows an example wherein 12 well units each having a couple ofwells connected via a channel as shown in FIG. 4 are mounted on a squaresubstrate 7 (16 mm×16 mm). In this example, the units are each 5.7 mm inthe major sides and 1.2 mm in the minor sides and located at intervalsof 0.8 mm.

FIG. 17 shows an example of an integration of multiplicity of integratedunits. In FIG. 17, each of quadrilaterals A₁₋₄, B₁₋₄ and C₁₋₄corresponds to the integration shown by FIG. 16. In this case, thearrays A, B and C are integrations of units of different types.

FIG. 18 shows an example wherein independent double system units areintegrated circularly. FIG. 19 is a sectional view of the unit of FIG.18 along the dashed and dotted line. Concerning the size, for example,the width of wells 2A and 2B in the radial direction is 1.5 mm, thewidth of a channel 1 in the redial direction is 0.5 mm and the width ofgrooves13 formed in the channel 1 is 10 μm. In this case, the radius ofthe whole unit is 5.0 mm.

FIG. 26 shows an example wherein 12 units of the type shown by FIG. 24are integrated.

In such a case of integrating multiplicity of units, a single block 7and a single glass substrate 6 may be used so as to cover the whole unit(see FIG. 20).

FIG. 20 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells and separating chemotactic cellscomprising multiplicity of units integrated together. A substrate 5having multiplicity of units integrated thereon, a packing 5′ and ablock 7 covering them are placed between a cover cap 17 and anintermediate base 21. A glass substrate 6 is placed between theintermediate base 21 and a bottom base 22 and fastened with screws. Thelocations of the block 7 and the substrate 5 are specified by theintermediate base 21 and fixed by guide pins 20 and guide pin receiverholes 19 provided at the bottom face of the block 7. Alternatively, thesubstrate 5 may be directly pressed and fixed to the block 7.

In FIG. 20, it is also possible that a substrate 5 having a single unit(i.e., a couple of wells and a channel) is used as a substitute for theintegrated unit and a plural number of the fabricated units are arrangedat definite intervals. In this case, units can be successivelyexchanged.

8) Automatic Controlling System

Next, the automatic controlling system in the microsample treatmentapparatus according to the present invention will be illustrated indetail by reference to an apparatus for detecting chemotaxis of cells asan example. However, it is needless to say that this illustration isgiven merely by way of example and various embodiments may be furtheremployed for achieving the automation.

FIG. 27 shows an example of the automatic controlling system of theapparatus for detecting chemotaxis of cells according to the presentinvention. In FIG. 27, U represents a unit part, C represents a cellreservoir, S represents a specimen reservoir and W represents a pipettewashing part. The line X-X′ shows an example of the flow line of aplural number of specimen supply pipettes (6 in this case) alignedlaterally, while the line Y-Y′ shows an example of the flow line of aplural number of cell supply pipettes aligned laterally. The unit part Uis set at the pipette flow line position and a space provided above thetop ends of each unit is filled up with a liquid. Cells are held in thecell reservoir, while various specimens are held in the specimenreservoir S. Liquid level control pipettes aligned laterally are locatedabove the unit part 4B to 4A and the flow line thereof is indicated by,for example, Z-Z′ in FIG. 28. Each pipette is moved, for example, asfollows, though it is needless to say that the present invention is notrestricted thereto.

A definite amount of a cell suspension is sucked from the cell reservoirC by a cell supply pipette. Then the pipette moves along the flow lineY-Y′ to the unit part U and supplies the cell suspension into the well2A of each unit through a cell supply tube 3A. Subsequently, the cellsupply pipette returns to the position C and stops the operation, ormoves to supply the cell suspension to the next unit. Since cells areprecipitated owing to the gravity, it is favorable to stir the cellsuspension contained in the cell-reserving container 25 immediatelybefore collecting the cells by sucking.

Next, the liquid in the space 10 in each unit is sucked by a liquidlevel control pipette and thus the liquid level is lowered to theposition II, as FIG. 28 shows. Subsequently, a definite amount of theliquid is further sucked so as to adjust the position of cells in thewell 2A. Then the liquid level control pipette is elevated to the liquidlevel I position or higher and the sucked liquid is discharged at anypoint on the flow line Z-Z, thereby returning the liquid level in thespace 10 to the position I. Subsequently, the liquid level controlpipette is further elevated and stops its operation, or moves on thenext unit.

Then a definite amount of a specimen is sucked from the specimenreservoir S by a specimen supply pipette. The specimen supply pipettemoves along the flow line X-X′ to the unit part U and supplies thespecimen into the well 2B through a specimen supply tube 3B.Subsequently, the specimen supply pipette moves along the flow line X-X′to the pipette washing part W wherein it is washed by repeatedly suckingand discharging a washing liquor in a washing tank. Then the pipette iselevated above the liquid level in the washing tank and stops itsoperation, or moves to the next unit part U to supply the specimen.

Next, the unit part U having the cell suspension and the specimen thussupplied moves in the direction indicated by an arrow→in FIG. 27 andstops at the position where the channel 1 agrees with the detectionpart. Thus, the conditions of the cells are detected and recorded. Asthe unit part U moves, the next unit part U comes to the position of thepipette flow line and thus the above operations are repeated. It is alsopossible to move the unit part U together with the specimen reservoir S.In this case, the unit part U and the specimen reservoir S movestogether and thus the next unit part U and the next specimen reservoir Scome to the pipette flow line.

The cell reservoir C is provided with containers for temporarily holdingcells to be supplied into the unit part U. These containers may be inany shape, so long as they can play the desired role. FIG. 29 shows anexample of the containers in the cell reservoir C. A plural number ofcell containers 25 are provide,d corresponding to the arrangement ofeach unit and a plural number of the cell supply pipettes. In theexample shown by FIG. 29, an inclined injection part 26 is formed tofacilitate the injection of cells into each container and avoid waste ofcells. It is preferable to further provide an inlet part 27 so that thecell suspension can be easily introduced into the containers withoutwaste. By using this structure, the cell suspension injected at anarbitrary point can be supplied into all containers, thereby saving alot of time and labor for injecting the cell suspension into individualcells. It is also preferable that the cell containers 25 are tapered atthe bottom so as to avoid waste of the cell suspension in the step ofsucking by the pipettes. In FIG. 29, (1) is a perspective view; (2) is atop plan view; (3) is a sectional view along the dotted line A-A′ in(2); and (4) is another sectional view along the dotted line B-B′ in(2).

The specimen reservoir S is provided with containers for temporarilyholding a specimen to be supplied into the unit part U. These containersmay be in any shape, so long as they can play the desired role. In caseof supplying many types of specimens into the unit part U, use isfrequently made of a method wherein individual specimens are manuallyinjected into the containers in the specimen reservoir S with the use ofmicropipettes, etc. In such a case, it is preferable to provide pipettetip inlet ports 29 having a diameter larger than the pore size of theopening of the containers, as shown in FIG. 30. It is also desirablethat the containers are tapered at the bottom to lessen the specimenremaining therein after taking out from the containers, as shown by FIG.30. In FIG. 30, (1) is a perspective view; (2) is a sectional view; and(3) is a top plan view. In the example shown by FIG. 30(2), the pipettetip 34 is inserted into the container 28 from the pipette tip inlet port29 in the step of manually injecting a specimen. FIG. 31 shows anexample wherein a plural number of specimen containers are located alongthe flow line X-X′ of the specimen supply pipette. As FIG. 31 shows, theinlet ports may be alternately located so that the intervals among thecontainers can be adjusted fit to the intervals among the units in theunit part U. The specimen containers may have a square shape, as shownby FIG. 32. FIG. 33 shows an example wherein a plural number of specimencontainers are located along the flow line X-X′ of the specimen supplypipettes.

In the pipettes to be used in the apparatus according to the presentinvention, suction and discharge of liquids can be controlled bycomputerized programming. It is preferable to use a pipette having amultichannel syringe as shown by FIG. 34. The needle (tip) of thepipette may be made of glass, a metal, a plastic material, etc. In FIG.34, (1) is a top plan view; and (2) is a side plan view.

The detection means to be used in the present invention may be any meansso long as cells which are passing through a channel or have passedtherethrough can be detected thereby. If necessary, it involves a meansof recording the detection data. Any means known as a means of detectingand recording cells is usable therefor. Use can be made of, for example,a microscope optionally combined with a video camera. It is alsopossible to employ a system having an objective lens provided with a CCDcamera. For the detection in integrated units, it is preferable toemploy a system wherein the channels of the units are successivelyscanned along with an objective lens.

As shown by FIG. 4, the detection means is usually provided in a channelof a unit. In an apparatus having multiplicity of units integratedtogether, it is also possible to employ a system wherein arrays of theunits successively come to a detection part located at a definiteposition for detection and recording. In this case, the channels of thealigned units are scanned with the detector. Either one or more scanningdetectors may be employed. Owing to this constitution, a relativelysmall number of detectors suffice for the detection in multiplicity ofintegrated units.

Cells which are passing or have passed through a channel can be detectedand counted by directly observing the cells with a microscope.Alternatively, the detection and counting can be easily performed bypreliminarily labeling the cells with a luminous or fluorescentsubstance and then capturing the luminescence or fluorescence in aconventional manner.

INDUSTRIAL APPLICABILITY

According to the structure of the present invention, it is possible to,in the step of injecting a liquid sample into a well, prevent themigration of the sample into another well or overflow thereof. Moreover,the position of the injected sample can be adjusted in a well or thesample can be transferred into the next well under controlling.

The structure according to the present invention achieves a remarkabletechnical merit and widely applicable particularly in cases of handlingsamples in microquantities such as solutions and cell suspensions, orseparating cells or particles depending on size.

A high technical merit can be established by applying the structure ofthe present invention to an apparatus for detecting chemotaxis of cellsor an apparatus for separating cells with the use of cell chemotaxis.That is to say, unexpected migration of a sample caused by pressurechanges in the step of injecting/sucking samples such as cells andspecimen solutions can be prevented thereby. Furthermore, unexpectedmigration of a sample caused by horizontal off balance of the apparatuscan be prevented. Thus, movements of cells by their own actions can beaccurately understood or desired cells can be taken out. Namely, it ispossible to obtain results affected by both of the effect of achemotactic factor or an inhibitor and the characteristics of the cells.

In the apparatus for detecting chemotaxis of cells or the apparatus forseparating cells with the use of cell chemotaxis according to thepresent invention, a bank is formed in a channel located between wellsor barriers constituting definite grooves are formed on the bank or,alternatively, a gap is formed between a plane provided on the upperface of the bank and a glass substrate. Owing to this structure, itbecomes possible to easily establish the state wherein cells are broughttogether in the vicinity of the channel and aligned in the flowdirection of the cells, when a cell suspension is put into one well andan adequate amount of a liquid is sucked from the other well. As aresult, the presence/absence of the cell chemotaxis can be accuratelydetected.

The structure according to the present invention makes it possible todownsize the apparatus. When applied to an apparatus for detectingchemotaxis of cells or separating chemotactic cells, namely, samples canbe used in an amount 1/50 to 1/1000 times as much in the conventionalcases with the use of a Boyden chamber. That is to say, biologicalsamples (whole blood, etc.) per se are usable as samples in theapparatus of the present invention. By using whole blood as a sample,for example, measurement can be made by using 0.1 μl of blood in case ofdetecting the chemotaxis of neutrophils and about 1 μl of blood in caseeosinophils, monocytes or basophils.

In the structure according to the present invention, moreover, nodelicate control is needed in the step of injecting a liquid, whichbrings about an additional merit that the apparatus can be easilyautomated.

The unit of the apparatus according to the present invention can be in amicrosize and thus multiplicity of the units can be integrated together,which brings about another merit that an apparatus whereby a largenumber of samples can be simultaneously treated can be fabricated. Inthis case, an apparatus having an automated system of injecting anddetecting liquids can be easily fabricated.

In integrating multiplicity of units, detection and separation fordifferent purposes can be simultaneously carried out by combining andintegrating units of different types together. Thus, the treatmentefficiency can be elevated. In case of an apparatus for detectingchemotaxis of cells, for example, the detection of various chemotacticfactors or inhibitors for a single type of cells or the detection of thechemotaxis of different types of cells for a single chemotactic factorcan be carried out at once.

1. A microsample treatment apparatus, which comprises: a) a plurality ofwells; b) said plurality of wells brine connected to each other via apart having resistance to fluids, said part being selected from thegroup consisting of one or more thin pipes, narrow gaps, narrow caphaving barriers, thin grooves, filters, resin-filled columns and otherstructures through which a fluid can be passed but which have resistanceto fluids; and c) each of the wells being provided with at least onetube for injecting/sucking a sample, wherein 1) said apparatus has aspace in common at the top ends of said tubes in which a liquid is heldto maintain said liquid in said wells and said part having resistance tofluids under a definite pressure, 2) the level of said liquid can becontrolled to adjust the position of said sample in said well(s) ortransfer said sample into the other well(s) across the part havingresistance, to fluids, and 3) the top end of at least one tube formed ina well is located higher than top ends of the tubes formed in one ormore wells across the part having resistance to fluids.
 2. Themicrosample treatment apparatus as claimed in claim 1, wherein each ofthe wells has a single tube.
 3. The microsample treatment apparatus asclaimed in claim 1, wherein each of the wells has a plurality of saidtubes.
 4. A microsample treatment apparatus which comprises: a) anintegration unit having a plural number of units of the same ordifferent types on a single substrate or a plural number of integrationunits on a single substrate; b) said unit which has a plural number ofwells; c) said plurality of wells being connected to each other via apart having resistance to fluids, said part being selected from thegroup consisting of one or more thin pipes, narrow gaps, narrow gaphaving barriers, thin grooves, filters, resin-filled columns and otherstructures through which a fluid can be passed but which have resistanceto fluids; and d) each of the wells being provided with at least onetube for injecting/sucking a sample, wherein 1) said unit has a space incommon at the top ends of said tubes in which a liquid is held tomaintain said liquid in said wells and said part having resistance tofluids under a definite pressure and 2) the level of said liquid can becontrolled to adjust the position of said sample in said well(s) ortransfer said sample into the other well(s) across the part havingresistance to fluids, and 3) the top end of at least one tube is formedin a, well located higher than top ends of the tubes formed in one ormore wells across the , part having resistance to fluids.
 5. Themicrosample treatment apparatus as claimed in claim 4, wherein each ofthe wells has a single tube.
 6. The microsample treatment apparatus asclaimed in claim 4, wherein each of the wells has a plurality of saidtubes.
 7. An apparatus for detecting chemotaxis of cells or separatingchemotactic cells, which comprises: a) a plurality of wells; b) saidplurality of wells being connected to each other via a channel having awidth fit for a diameter or deformability of cells; and c) each of thewells being provided with at least one tube for injection/sucking asample, wherein 1) said apparatus has a space in common at the top endsof said tubes in which a liquid is held to maintain said liquid in saidwells and said channel under a definite pressure, 2) the level of saidliquid can be controlled to adjust the position of the cells in saidwell(s), and 3) the top end of at least one tube formed in well forholding cells is located higher than top of the formed in one or morewells across the channel.
 8. The apparatus for detecting chemotaxis ofcells or separating, chemotactic cells as claimed in claim 7, whereineach of the wells has a single tube.
 9. The apparatus for detectingchemotaxis of cells or separating chemotactic cells as claimed in claim7, wherein each of the wells has a plurality of said tubes.
 10. Anapparatus for detecting chemotaxis of cells or separating chemotacticcells which comprises: a) an integration unit having a plural number ofunits of the same or different types on a single substrate or a pluralnumber of integration units on a single substrate; b) said each unit hasa plural number of wells; c) said plural number of wells being connectedto each other via a channel having a width fit for a diameter ordeformability of cells; and d) each of the wells being provided with atleast one tube for injecting/sucking a sample, wherein 1) said unit hasa space in common at the top ends of said tubes in which a liquid isheld to maintain said liquid in said wells and said channel under adefinite pressure, 2) the level of said liquid can be controlled toadjust the position of the cells in said well(s), and 3) the top end ofat least one tube formed in a well for holding cells is located higherthan top ends of the tubes formed in one or more wells across thechannel.
 11. The apparatus for detecting chemotaxis of cells orseparating chemotactic cells as claimed in claim 10, wherein each of thewells has a single tube.
 12. The apparatus for detecting chemotaxis ofcells or separating chemotactic cells as claimed in claim 10, whereineach of the wells has a plurality of said tubes.